Skeletal myoblasts are a population of progenitor cells in adult skeletal muscle that are capable of differentiating intovarious types of skeletal muscle. This unique capability of myoblasts makes them suitable candidates for tissue engineering applications such from cellular cardiomyoplasty and patches to replace damaged skeletal or cardiac tissue. Control of the differentiation and mechanical behavior of the cells is crucial to using these cells to repair damaged tissues. The use of a patch in which cells are grown in three-dimensional culture within a support material offers several advantages over direct injection of cells. The three-dimensional culture can be preconditioned to permit adaptation of cells to a specific mechanical environment and reducing apoptosis. Long-term objectives of our research are to determine the effect of mechanical stimulation upon skeletal muscle differentiation and function in order to design engineering replacements for damaged muscle tissue. In this project we propose to examine the hypotheses that (1) static and dynamic stimulation of myoblasts regulates the differentiated state of the skeletal muscle and (2) that mechanical stimulation regulates differentiation by coordinated interaction between nitric oxide synthase activity and cytoskeletal interactions with focal contacts. Our preliminary data show that nitric oxide release by mechanical stimulation accelerates differentiation and alters the mechanical properties of the cells. In order to address these hypotheses we will use the mouse myoblast cell line C2C12 which are mechanically stimulated in two- and three dimensional cultures. Two-dimensional cultures permit a detailed study of mechanical behavior and protein expression of the differentiating cells. Three-dimensional culture studies enable us to extrapolate the results to conditions in a realistic tissue engineering application. Specific aims of the proposed work are: (1) Evaluate the effect of static and dynamic mechanical stimulation on murine myoblast differentiation; (2) Determine the effect of type of mechanical stimulation upon the expression of nitric oxide synthase and NO release; (3) Examine the role of nitric oxide upon focal contact formation and interaction with the cytoskeleton; and (4) Evaluate the response of three-dimensional myoblast cultures to mechanical stimulation and the importance of nitric oxide delivery. Cells will be exposed to static and oscillatory stretch conditions that mimic conditions to which skeletal and cardiac muscle cells are exposed. We will examine the elastic and viscous behavior of the cells and the extent to which the behavior is influenced by nitric oxide release and nitric oxide synthase expression. Focal contact formation and actin filament organization will be assessed under different stretch conditions and the influence of focal contact formation on mechanical properties determined. These studies will provide important new data on the role of mechanical preconditioning on the differentiation of skeletal muscle myoblasts.